MIR16, a putative membrane glycerophosphodiester phosphodiesterase, interacts with RGS16

Abstract

We have identified the protein MIR16 (for Membrane Interacting protein of RGS16) from a yeast two-hybrid screen by using RGS16 as bait. MIR16 shares strong homology with bacterial glycerophosphodiester phosphodiesterases. It interacts with RGS16 and, more weakly, with several other selected RGS proteins. Analysis of deletion mutants showed that the N-terminal region of the RGS domain in RGS16 is required for its interaction with MIR16. MIR16 is an integral membrane glycoprotein, because it remained associated with membrane fractions after alkaline treatment and because, in some cells, it is sensitive to digestion with endoglycosidase H. By immunofluorescence and immunoelectron microscopy, MIR16 was localized on the plasma membrane in liver and kidney and on intracellular membranes in rat pituitary and cultured pituitary cells. MIR16 represents the only integral membrane protein identified thus far to interact with an RGS domain and, to our knowledge, is the only mammalian glycerophosphodiester phosphodiesterase that has been cloned. The putative enzymatic activity of MIR16 and its interaction with RGS16 suggest that it may play important roles in lipid metabolism and in G protein signaling.G protein ͉ integral membrane protein ͉ lipid metabolism H eterotrimeric G proteins transduce a variety of extracellular signals from G protein-coupled receptors to downstream G protein effectors (1, 2). Recently, a protein family, the RGS proteins (for Regulator of G protein Signaling), has been discovered; these proteins regulate G␣ subunits by acting as GTPase activating proteins (3-5). To date, at least 24 distinct RGS proteins have been found in mammals, but information on the connections of RGS proteins to specific signaling pathways and on the functional roles of RGS proteins in specific biological systems is still limited (3, 4).We have been interested in defining the G protein-mediated signaling pathways associated with intracellular membranes (6, 7). Previously, we identified GAIP (G␣ interacting protein), one of the founding members of the RGS protein family, and localized it to clathrin-coated vesicles in pituitary cells and hepatocytes, among others (6). Besides GAIP, additional RGS proteins have also been found in pituitary, liver, and other secretory cells (3). We have chosen RGS16 (ref. 8; ref. 9; ref. 10) for further study, because our interests focus on secretory cells and because RGS16 was shown to be highly expressed in mouse pituitary and liver (8). RGS16 has been shown to be a GTPase activating protein for members of the G␣i family in vitro (9, 11). However, little information is available concerning the interaction of RGS16 with molecules other than G␣ subunits.To define further signaling pathways involving RGS16, we used the yeast two-hybrid system to identify proteins that interact with RGS16. Herein, we report the discovery of an integral membrane protein MIR16 (for Membrane Interacting protein of RGS16), identified by screening a pituitary cell cDNA library with RGS16 as bait. Sequence analysis ind...

Anandamide Biosynthesis Catalyzed by the Phosphodiesterase GDE1 and Detection of Glycerophospho-N-acyl Ethanolamine Precursors in Mouse Brain

“…5B). Treatment with the glycosidase PNGase F reduced the apparent molecular mass of GDE1 by ϳ5 kDa, indicating that, as previously reported, the enzyme is an N-linked glycoprotein (19). These data are consistent with hydropathy plot analyses that predict the enzyme is an integral membrane protein with the majority of the protein sequence, including the catalytic domain, facing the luminal/extracellular compartments of the cell.…”

“…Among the five GDEs tested, one enzyme, GDE1, exhibited robust activity with GP-NAE. GDE1 was originally identified in a yeast two-hybrid screen for proteins that interact with RGS16, a regulator of G-protein signaling (19). Based on its interactions with RGS16, GDE1 is also referred to as MIR16 (for membrane interacting protein of RGS16).…”

Familial juvenile hyperuricaemic nephropathy (FJHN): linkage analysis in 15 families, physical and transcriptional characterisation of the FJHN critical region on chromosome 16p11.2 and the analysis of seven candidate genes

“…This alignment provides two genetic intervals -D16S499-D16S3036 (FJHN1) and D16S412-D16S3116 (FJHN2) delimited by critical recombinations detected in Belgian 11 and Japanese 33 families. From eight haplotypes 39 Sequence analysis showed heterozygous transition c1305A-G located in 3'UTR in one of the patients. This polymorphism was reported in dBSNP previously.…”

Mammalian Glycerophosphodiester Phosphodiesterases

“…Zheng et al used a yeast two-hybrid system to define the RGS16 (for regulator of G protein signaling) mediated signaling pathway, and isolated GDE1, a membrane protein interacting with RGS16. 8) In contrast, we employed a…”